Stroke, defined as a sudden weakening or loss of consciousness, sensation and voluntary motion caused by rupture or obstruction of an artery of the brain, is the third cause of death in the United States. Worldwide, stroke is the number one cause of death due to its particularly high incidence in Asia. Ischemic stroke is the most common form of stroke, being responsible for about 85% of all strokes, whereas hemorrhagic strokes (e.g. intraparenchymal or subarachnoid) account for the remaining 15%. Due to the increasing mean age of the population, the number of strokes is continuously increasing. Because the brain is highly vulnerable to even brief ischemia and recovers poorly, primary prevention in ischemic stroke prevention offers the greatest potential for reducing the incidence of this disease.
Focal ischemic cerebral infarction occurs when the arterial blood flow to a specific region of the brain is reduced below a critical level. Cerebral artery occlusion produces a central acute infarct and surrounding regions of incomplete ischemia (sometimes referred to as ‘penumbra’), that are dysfunctional-yet potentially salvageable. Ischemia of the myocardium, as a result of reduced perfusion due to chronic narrowing of blood vessels, may lead to fatal heart failure and constitutes a major health threat. Acute myocardial infarction, triggered by coronary artery occlusion, produces cell necrosis over a time period of several hours. In the absence of reflow or sufficient perfusion, the cerebral or myocardial ischemic regions undergo progressive metabolic deterioration, culminating in infarction, whereas restoration of perfusion in the penumbra of the brain infarct or in the jeopardized but salvageable region of the myocardium may ameliorate the tissue damage.
Growth factor mediated improved perfusion of the penumbra in the brain or of the jeopardized myocardium of patients suffering ischemic events, either via increased vasodilation or angiogenesis (the formation of endothelial-lined vessels), may be of great therapeutic value according to Isner et al. in J. Clin. Invest. (1999) 103(9):1231–6 but many questions yet remain to be answered in this respect. This is also true for ischemic conditions related to peripheral limbs (eg peripheral arterial disease, peripheral ischemia). For instance, an outstanding question is whether formation of new endothelial-lined vessels (i.e. angiogenesis) alone is sufficient to stimulate sustainable functional tissue perfusion. Indeed, coverage of endothelial-lined vessels by vascular smooth muscle cells (i.e. arteriogenesis) provides vasomotor control, structural strength and integrity and renders new vessels resistant to regression.
Capillary blood vessels consist of endothelial cells and pericytes, which carry all the genetic information required to form tubes, branches and entire capillary networks. Specific angiogenic molecules can initiate this process. A number of polypeptides which stimulate angiogenesis have been purified and characterized as to their molecular, biochemical and biological properties, as reviewed by Klagsbrun et al. in Ann. Rev. Physiol. (1991) 53:217–239 and by Folkman et al. in J. Biol. Chem. (1992) 267:10931–4. One factor that can stimulate angiogenesis and which is highly specific as a mitogen for vascular endothelial cells, is termed vascular endothelial growth factor (hereinafter referred as VEGF) according to Ferrara et al. in J. Cell. Biochem. (1991) 47:211–218. VEGF is also known as vasculotropin. Connolly et al. also describe in J. Biol. Chem. (1989) 264:20017–20024, in J. Clin. Invest. (1989) 84:1470–8 and in J. Cell. Biochem. (1991) 47:219–223 a human vascular permeability factor that stimulates vascular endothelial cells to divide in vitro and promotes the growth of new blood vessels when administered into healing rabbit bone grafts or rat corneas. The term vascular permeability factor (VPF for abbreviation) was adopted because of increased fluid leakage from blood vessels following intradermal injection and appears to designate the same substance as VEGF. The murine VEGF gene has been characterized and its expression pattern in embryogenesis has been analyzed. A persistent expression of VEGF was observed in epithelial cells adjacent to fenestrated endothelium, e.g. in chloroid plexus and kidney glomeruli, which is consistent with its role as a multifunctional regulator of endothelial cell growth and differentiation as disclosed by Breier et al. in Development (1992) 114:521–532. VEGF shares about 22% sequence identity, including a complete conservation of eight cysteine residues, according to Leung et al. in Science (1989) 246:1306–9, with human platelet-derived growth factor PDGF, a major growth factor for connective tissue. Alternatively spliced mRNAs have been identified for both VEGF and PDGF and these splicing products differ in their biological activity and receptor-binding specificity. VEGF is a potent vasoactive protein that has been detected in and purified from media conditioned by a number of cell lines including pituitary cells, such as bovine pituitary follicular cells (as disclosed by Ferrara et al. in Biochem. Biophys. Res. Comm. (1989) 161:851–858 and by Gospodarowicz et al. in Proc. Natl. Acad. Sci. USA (1989) 86: 7311–5), rat glioma cells (as disclosed by Conn. et al. in Proc. Natl. Acad. Sci. USA (1990) 87:1323–1327) and several tumor cell lines. Similarly, an endothelial growth factor isolated from mouse neuroblastoma cell line NB41 with an unreduced molecular mass of 43–51 kDa has been described by Levy et al. in Growth Factors (1989) 2:9–19.
Recent data show that VEGF has a modest effect on collateral growth and limb perfusion measured by microspheres (Deindl et al. in Circ. Res. (2001) 89:779–86), but increases perfusion measured by laser doppler (Isner et al. in Circulation (1999) 99:3188–98). Thus, despite a slight increase in collateral growth and hind limb perfusion, the overall functional reserve after VEGF treatment was impaired, presumably because of unfavorable hemodynamic effects. In other words, it is known that despite vessel formation by means of VEGF, the functionality of the vessels formed is very low. Despite promising initial clinical trials, a large double-blind placebo-controlled trial testing the efficacy of intracoronary and intravenous VEGF for therapeutic angiogenesis in patients with chronic myocardial ischemia not amenable to standard revascularization techniques, indicated that even high dose treatment with VEGF did not lead to a significant improvement in ETT time (Henry et al. 2003, Circulation, 107:1359–1365).
Placental growth factor (hereinafter referred as PlGF) was first disclosed by Maglione et al. in Proc. Natl. Acad. Sci. USA (1991) 88(20):9267–71 as a protein related to the vascular permeability factor. U.S. Pat. No. 5,919,899 discloses nucleotide sequences coding for a protein, named PlGF, which can be used in the treatment of inflammatory diseases and in the treatment of wounds or tissues after surgical operations, transplantations, burns of ulcers and so on. Soluble non-heparin binding and heparin binding forms, built up of 131 and 152 amino-acids and known as PlGF-1 and PlGF-2 respectively, have been described for PlGF and were found to be differentially expressed in placenta, trophoblastic tumors and cultured human endothelial cells (Maglione et al., 1993).
Carmeliet et al. Nat. Med. (2001) 7:575–583 provides an extensive and comprehensive review of the state of the art related to PlGF as follows. VEGF stimulates angiogenesis by activating the VEGF tyrosine kinase receptor-2 (VEGFR-2). Recombinant PlGF stimulates angiogenesis in particular conditions and induces vascular permeability when co-injected with VEGF, but the role of endogenous PlGF remains unknown. Both PlGF and VEGF bind to VEGF receptor-1 (VEGFR-1). PlGF has been proposed to stimulate angiogenesis by displacing VEGF from the ‘VEGFR-1 sink’, thereby increasing the fraction of VEGF available to activate VEGFR-2. Alternatively, PlGF might stimulate angiogenesis by transmitting intracellular signals through VEGFR-1. PlGF might also affect angiogenesis by forming heterodimers with VEGF, but their role is controversial.
The role of PlGF in angiogenesis was evaluated by inactivating the gene expressing PlGF (Pgf) in mice. Unexpectedly, the absence of PlGF had a negligible effect on vascular development. However, PlGF deficiency reduced pathological angiogenesis, permeability and collateral growth in ischemia, inflammation and cancer.
Carmeliet et al. (cited supra) studied the growth of collateral arteries after ligation of the femoral artery in mice and found that PlGF levels were 45% higher in ligated vessels than in control vessels. On the other hand macrophages, known to play an essential role in collateral growth, infiltrated significantly more collaterals in wild-type mice than in Pgf−/− mice. In another experiment, mice were transplanted with congenic bone marrow and capillary ingrowth in matrigel, supplemented with the VEGF165 isoform, and angiogenesis was quantified by measuring the hemoglobin content per implant. Matrigel angiogenesis was abundant when both the donor bone marrow and the host vessels produced PlGF but minimal when the bone marrow and host lacked PlGF. Capillaries still infiltrated the matrigel in wild-type mice transplanted with Pgf−/− bone marrow, indicating that PlGF production by vessel-wall-associated endothelial cells was sufficient for angiogenesis. When a Pgf−/− recipient was transplanted with wild-type bone marrow, matrigel angiogenesis still occurred, indicating that production of PlGF by bone-marrow cells can stimulate angiogenesis at distant sites. Transplantation of wild-type bone marrow in Pgf−/− mice also rescued the impaired collateral enlargement after ligation of the femoral artery, possibly by mobilizing PlGF-producing monocytes/macrophages to the collaterals. In still another experiment, in order to determine whether the amplification of the VEGF response by PlGF resulted from direct stimulation of endothelial cells, Carmeliet et al. (cited supra) studied capillary outgrowth in intact aortic rings and found that, because PlGF alone (in the absence of VEGF) did not stimulate isolated endothelial cells, PlGF probably stimulated capillary outgrowth by amplifying endogenous VEGF in aortic rings. Carmeliet et al. (cited supra) further observed that even at low doses PlGF dose-dependently restored the impaired VEGF response of Pgf−/− endothelial cells. Further, PlGF activated VEGFR-1. These findings altogether indicate that PlGF, via activation of VEGFR-1, specifically potentiates the angiogenic response to VEGF and that by affecting vascular growth and remodeling, PlGF contributes to the pathogenesis of several disorders with high morbidity.
One problem to be solved by the present invention is to provide a pharmaceutical composition and methods particularly suited for improving perfusion of tissues of patients suffering ischemic events, which will prove to be useful for the prevention and treatment of strokes and ischemic diseases. Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods for reducing or suppressing infarct expansion of the penumbra during ischemic cerebral infarction and for enhancing revascularization of acute myocardial infarcts, making them useful for preventing and treating such events. Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods for enhancing revascularization of tissues in limb-threatening ischemia making them useful for treating such a disease.
Another problem to be solved by the present invention is to provide pharmaceutical compositions and methods not only for enhancing vascularization but for restoring the functionality of ischemic tissue, in particular ischemic muscles such as the cardiac muscle and skeletal muscles in the head, neck, thorax, abdomen, back and upper and lower limbs. There is also a need in the art for curing a reduced physical muscular performance, especially that of heart and the aforesaid muscles after an ischemic event in a mammal, in particular a human being. Another problem to be solved by the present invention is providing a pharmaceutical and methods which can be used as described above without incurring the well known disadvantages of some growth factors such as VEGF, e.g. hypotension after systemic administration of high doses over short periods of time (see for instance Hariawala et al. in J. Surg. Res. (1996) 63(1):77–82).